Fishing to Design Inherently Safer Nanoparticles - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 280

20.4.4 Cellular Death

The embryonic zebrafish model is ideal for mapping cellular death in the whole
animal and identifying the mechanism of cell death (i.e., necrosis versus apoptosis).
To determine overall cellular death, embryos are exposed to nanoparticle solutions as
described for tier 1, and then stained with acridine orange to label cells that have lost
cell membrane integrity. The terminal deoxynucleotidyl transferase (TdT)-mediated
dUTP nick end labeling (TUNEL) assay is used specifically to quantify apoptosis.

20.4.5 In Vivo Nanoparticle Distribution

Determining nanoparticle localization, even in an organism as small as larval
zebrafish, is technically challenging. Distribution of nanoparticles assessed in tier
1 and 2 testing is currently tracked in two ways. The simpler approach is to
fluorescently label the nanoparticle. In the case of a waterborne exposure, fluorescent
microscopy can then be used to determine whether the nanoparticle was absorbed or
was simply stuck to the epithelium. This simple discrimination cannot be determined
using INAA, ICP-OES, and ICP-MS methods described in tier 1. A caveat to the
fluorescently labeled approach is that the label could alter the nanoparticle properties
altering the uptake profile compared to the native nanoparticle.
The second method for detecting nanoparticle distribution is to use high-
resolution imaging scanning emission microscopy (SEM) or transmission electron
microscopy (TEM) to scan throughout the animal. To do this, nanoscale slices are cut
through mounted embryos and images are acquired using the electron microscopes.
This is followed by the use of software to overlay adjacent images, thereby recreating
a three-dimensional picture of the whole animal. Distribution data are critical to
understanding the mechanism of how each nanoparticle interacts with the biological
system. Developing nanoparticles for biomedical application must be accompanied
by careful assessment of their biodistribution for the product to be both efficacious
and safe.
Data gathered on the localization of oxidative stress, cell death, and distribution
of nanoparticles in tier 2 are processed into the NBI knowledge base and combined
with the data gathered from tier 1. This accumulation of data for each nanoparticle
enables conclusions to be drawn about structure-activity relationships in tier 3.

20.5 TIER 3: MOLECULAR EXPRESSION

Once distribution is localized, the nanoparticle is moved to tier 3 where global gene
expression profiling is conducted following waterborne exposures that elicit a tier 1
effect in 100%of the subjects (i.e., EC 100 ). Gene expression patterns, coupled with the
nanoparticle distribution data, can then be used to help identify cellular targets. For
example, if the nanoparticles are located in the liver, then onemight reasonably expect
to see altered expression of liver-specific genes as an activity resulting from the
exposure. There are various aspects that must be considered to conduct a useful global